Project Summary In patients suffering from malignancy, tumor-reactive T cells are present in tumor sites but have lost their ability to kill tumor cells due to a dysfunctional state termed ?exhaustion?. Exhausted T cells are characterized by their inhibition along two axes of function: the Checkpoint Receptor Axis, with overexpression of inhibitory receptor molecules such as PD1, Tim3, and Lag3; and the Cytokine Axis, with suppression of production of Type I cytokines, most prominently interleukin 2 (IL2). The Checkpoint Receptor Axis has attracted the most focus in clinical settings. FDA has approved anti-PD1 antibody drugs, Opdivo [nivolumab] and Keytruda [pembrolizumab] that mediate Checkpoint Receptor Blockade (CRB) for the treatment of metastatic melanoma, non-small cell lung cancer and other tumors. Still, only 20% to 40% of patients with these cancers respond to CRB therapy, with >50% of patients obtaining no benefit, indicating considerable room for improvement. We hypothesize, because anti-PD1 drugs treat only the Checkpoint Receptor Axis and not the Cytokine Axis, that CRB reversal of exhaustion is less than complete. We further hypothesize that the CRB experience may underrepresent the responses that are possible with a more complete reversal of exhaustion that also addresses the Cytokine Axis concurrently. Our research recently discovered that transcription factor YY1 is master regulator of T cell exhaustion, mediating both the upregulation of checkpoint receptors (Checkpoint Receptor Axis) and the suppression of Type I cytokines (Cytokine Axis) (Balkhi et al, iScience 2018;2:105-22). We show that small molecule inhibitors of Ezh2 are capable of fully reversing the IL2 failure under the Cytokine Axis. With these new discoveries, a perspective is enabled of combining CRB with Ezh2 inhibitor, addressing both Axes concurrently as envisioned in our Hypotheses above, for a more complete reversal of exhaustion and improved clinical responses. To date, our data are confined to the human system. The goal of this project is to confirm these mechanisms in mouse T cells and to perform in vivo testing: i.e., to determine if Ezh2 inhibition will synergize with CRB to better restore function to exhausted T cells for superior anti-tumor activity over CRB alone. If our Hypothesis is confirmed, human clinical trials will follow from which we anticipate Ezh2 inhibitors will be approved as combination therapy in conjunction with CRB with anti-PD1 immunotherapy drugs.